Inhibitory effects of Zanthoxylum rhoifolium Lam . ( Rutaceae ) against the infection and infectivity of macrophages by Leishmania amazonensis

Zanthoxylum rhoifolium Lam. (Rutaceae) has been traditionally used in the treatment of microbial infections and parasitic diseases. In the present study, the antileishmanial effect induced by the ethanol extract of stem barks from Z. rhoifolium (ZR-EEtOH) and its n-hexane fraction (ZR-FHEX) on infection and infectivity of murine macrophages by promastigote forms of Leishmania amazonensis were investigated. In different set of experiments, macrophages or promastigotes were pretreated with ZR-EEtOH or ZR-FHEX at non-lethal concentrations for 24 hours, and then macrophages were submitted to infection by promastigotes. Moreover, their effects on activation of macrophages, as well as on the DNA content, size and number of promastigotes by flow cytometry were also evaluated. The infection rate and the number of internalized amastigote forms were markedly decreased after pretreatment of macrophages or promastigotes when compared with nontreated cells. The increase in phagocytic capability and nitrite content was also observed. Furthermore, the decrease of DNA content, size and number of promastigotes was also observed. In conclusion, ZREEtOH and ZR-FHEX promoted a markedly significant antileishmanial effect and reduction of infection of macrophages, probably underlying defense mechanisms activation in macrophages. These findings reinforce the potential application of Z. rhoifolium in the treatment of leishmaniasis.


IntRoductIon
The leishmaniasis is a complex of diseases caused by digenetic protozoa from Leishmania genus which affects around two million people per year and is also considered an endemic disease in 88 countries.Furthermore, around 350 million of people are under the risk throughout the world (Monzote et al. 2007, World Health Organization 2010).The treatment of this disease is based on drugs which possess a sort of drawbacks, as follows: only parenteral administration is possible,
higher toxicity, high costs, long-lasting therapy and therapeutic spectrum restricted to only specific clinical forms of the disease (Chappuis et al. 2007, Lindoso et al. 2012).Traditionally, medicinal plants have been widely used in traditional medical practices to treat parasitic diseases and currently have received special attention concerning the search for new therapies against leishmaniasis.Therefore, several plants and their respective isolated compounds, such as chalcones, lignans, alkaloids, flavonoids, terpenes, saponins and quinones, have been studied for the treatment of leishmaniasis and have shown to be promising antileishmanial agents (Kayser et al. 2001, Sen and Chatterjee 2011, Singh et al. 2014).Zanthoxylum rhoifolium Lam.(Rutaceae) is a widespread plant that occurs in South America, especially in Brazilian rainforest, in the states of Minas Gerais, Rio de Janeiro, Piauí and Ceará (Freitas et al. 2011, Gonzaga et al. 2003, Pereira et al. 2010).This species is traditionally used in the treatment and prevention of malaria.In Bolivia, the bark and leaves of Z. rhoifolium are used as antipyretic.In Peru, it is used due to its digestive and tonic properties.Besides, Z. rhoifolium is popularly used in Brazil for treatment of inflammation, microbial infection, cancer and malaria (Jullian et al. 2006, Da Silva et al. 2007).Furthermore, a sort of secondary metabolites, mainly lignans, alkaloids, terpenoids and flavonoids, have been found in this species.Likewise, studies have shown that these alkaloids exhibit antimicrobial properties (Gonzaga et al. 2003, Tavares et al. 2014).
Previous reports have demonstrated antinociceptive (Pereira et al. 2010), gastroprotective (Freitas et al. 2011) and antihypertensive effects (Ferreira-Filho et al. 2013) for Z. rhoifolium in different rodents experimental models.Interestingly, a preliminary study reports the leishmanicidal effect for Z. rhoifolium against promastigote forms of Leishmania amazonensis (Moura-Costa et al. 2012).However, its effects on the ability of L. amazonensis to infect macrophages as well as its ef-fects against internalized amastigote forms of L. amazonensis remain still unknown.Therefore, the antileishmanial effects of Z. rhoifolium against infection and infectivity of macrophages by L. amazonensis, as well as the possible underlying mechanisms, were evaluated in this study.

botanical Source, eXtractS and fractioning
Stem barks from Z. rhoifolium Lam.(Rutaceae) were collected in January 2005 at Pedro II city, Piauí state, Brazil.The specimens were trees around 10 m of height and their stems were around 29.5 cm of diameter.The pluviometric index was around 1150.2 mm and the predominant soil was dystrophic red-yellow latosol (oxisol).The botanical identification was performed by Profa.Dra.Roseli Farias Melo de Barros.The voucher specimen (TEPB 13870) was deposited at Graziela Barroso Herbarium of Federal University of Piauí (UFPI).Stem barks of Z. rhoifolium (1.0 kg) were dried, powdered and then extracted exhaustively at room temperature with ethanol.The solvent was removed by rotaevaporation, yielding the ethanol extract (ZR-EEtOH 85.0 g; 8.5%, w/w).Then, the ZR-EEtOH (50 g) was fractioned in n-hexane, yielding ZR-FHEX (7.0 g; 14.0%, w/w).Both ZR-EEtOH and ZR-FHEX were lyophilized.For the experimental protocols, they were dissolved in dimethylsulfoxide (DMSO) to a maximum concentration of 0.5%.
From ZR-FHEX, the pentacyclic triterpenoid lupeol was isolated by fractionation in a silica gel column, with gradient elution from n-hexane-ethyl acetate (95:5), providing a solid material which was crystallized with methanol and indentified as lupeol (1.1 g; 12.5%, w/w) by determination of its structure by NMR analysis (Pereira et al. 2010).
animalS Male or female Swiss mice (20-25 g, 6 weeks) were used for obtaining peritoneal macrophages.
All procedures involving animal experimentation were according to the Brazilian Law no.1,000 of 2012, and previously approved by the Animal Experimentation Ethical Committee from the Federal University of Piauí, Brazil (0022/2010).
aSSeSSment of cytotoXic activity of Z. rhoifolium Thioglycollate (3%; 1.5 mL) was administered in the peritoneal cavity of Swiss mice.After 72 hours, the macrophages were elicited by washing the abdominal cavity with 8 mL of sterile phosphate buffered saline (PBS) at pH 7.4 and 4 °C.The aspirate was centrifuged at 4 °C and 184 G for 10 minutes, resuspended in RPMI 1640 medium and the cells plated in 24-well plates (110 5 cells/500 µL of RPMI 1640 medium).Afterwards, the medium was replaced by RPMI 1640 supplemented with 10% FCS, penicillin and streptomycin, and then ZR-EEtOH or ZR-FHEX (100 to 3.12 μg/mL) were incubated for 48 hours at 5% CO 2 and 37 ºC.
The cell viability was based on capability of cells to reduce -2,5diphenyltetrazoliumbromide) to formazan.After 4 h, the absorbances were readed at 550 nm (Reilly et al. 1998, Carneiro et al. 2012).
effect of Z. rhoifolium on infection by L. amazonensis PromaStigoteS after Pretreatment of macroPHageS Macrophages were cultured as described above.After cell adhesion, the medium was replaced by RPMI 1640 supplemented with 10% FCS, penicillin and streptomycin.Then, 88.6,and 22.1 µg/mL) or ZR-FHEX (80, 20, and 5 µg/ mL) was added.After 30 minutes, the medium was replaced again and then 210 6 infective promastigotes were added per well.After 3 hours, the coverslips were removed and stained with Giemsa.For each coverslip, 100 cells were assessed, and the numbers of infected macrophages and internalized amastigotes per macrophage were counted as described by Soares et al. (2007).Three independent experiments were carried out in triplicate for each concentration.

effect of Z. rhoifolium on tHe infective caPability of L. amazonensis PromaStigoteS
The macrophages were elicited as described above, and then plated at 210 5 per well in sterile 96-well plates with RPMI 1640 containing sterile 13 mm round coverslips.After cell adhesion, the medium was replaced by RPMI 1640 supplemented with 10% FCS, penicillin and streptomycin.Then, promastigotes were incubated at 26 ºC with ZR-EEtOH or ZR-FHEX at their respective 24-h half mean inhibitory concentrations (1/2 IC 50 ) for 30 minutes.Then, the promastigotes were centrifuged (367 G, 4 °C) for 10 minutes, resuspended in RPMI 1640 and plated at ratio of 10 promastigotes per macrophage.After incubation at 37 °C and 5% CO 2 for 3 hours, the coverslips were removed and stained with Giemsa.For each coverslip, 100 cells were assessed, and the numbers of infected macrophages BERNARDO MELO NETO et al. and internalized amastigotes per macrophage were counted as described by Soares et al. (2007).Three independent experiments were carried out in triplicate for each concentration.Association Indexes (AI) were obtained by the mean number of internalized amastigotes multiplied by the percentage of infected macrophages (Rosa et al. 2003).

determination of PHagocytic caPability and lySoSomal volume
Macrophages were obtained and plated (210 5 per well) as previously described.After cell adhesion, the medium was replaced by RPMI 1640 supplemented with 10% FCS, penicillin and streptomycin.Then, ZR-EEtOH or ZR-FHEX (100 to 3.12 μg/mL) were incubated for 48 hours at 5% CO 2 , 37 ºC.For determination of phagocytic capability, 10 µL of stained zymosan solution (neutral red solution 0.3 mL and zymosan not opsonized 0.02 g in PBS 3 mL) was used.Besides, for determination of lysossomal volume, 10 µL of 2.0% neutral red solution in DMSO was used.Both solutions were separately incubated for 30 min at 5% CO 2, 37 °C.The supernatants were discarded and 100 mL of an extractive aqueous solution composed by 96% glacial acetic acid (1%, v/v) and ethanol (50%, v/v) was added for 30 minutes.The absorbances were readed at 550 nm (Bonatto et al. 2004).
eXtraction, Purification and quantification of dna from L. amazonensis after Pretreatment witH Z. rhoifolium Promastigote forms of L. amazonensis at logarithmic growth phase (1×10 5 ) were pretreated with ZR-EEtOH or ZR-FHEX at concentrations of 7.96, 15.92 and 31.84μg/mL for 72h.Afterwards, the DNA from parasites was extracted by using the QIAamp® DNA MiniKit (QIAGEN, Venlo, Netherlands).Briefly, the parasites were centrifuged at 184 G, the pellets were resuspended with 200 μL of PBS, 20 μL of proteinase K, 4 μL of RNAse and 200 μL of AL Buffer were added and incubated at 56 ºC for 30 min.Then, 200 μL of ethanol (96%) was added, the samples were transfered to a 2.0 mL column and centrifugated (6.000 G for 1 minute).Afterwards, a sequence of buffers was sequentially added and followed by centrifugation, as follows: 500 μL of AW1 buffer, 6,000 G for 1 minute; 500 μL of AW2 buffer, 20,000 G for 4 minutes; and 500 μL of AE buffer, 6.000 G for 1 minute (manufacturer's protocol).Then, the DNA samples were quantified by a NanoDrop 2000 UV-Vis spectrophotometer (Thermo Scientific, Wilmington, DE, USA).
Moreover, the DNA samples from L. amazonensis pretreated with ZR-EEtOH or ZR-FHEX were also submitted to enzymatic digestion by Eco RI e Hind III enzymes (Sigma-Aldrich, St. Louis, MO, USA).Briefly, 2 μL of buffer and 6 μL of samples were incubated in a MasterCycler ® gradient thermal cycler at 37 ºC for 120 minutes (Eppendorf AG, Hamburg, Germany).These preparations were submitted to an inactivation process at 65 ºC for 20 minutes, followed by a running in gel electrophoresis in 1.5% agarose gel (25 V, 300 mA) for 120 minutes.According to the previous section, promastigote forms of L. amazonensis were evaluated by growth and viability after pretreatment with ZR-EEtOH or ZR-FHEX for 24h, 48h and 72h.Afterwards, the viable cells previously treated at concentration of 7.96 mg/mL were submitted to flow cytometry analysis by BD-FACSCanto® II cell analyzer (BD Company, Franklin Lakes, NJ, USA).A total of 10,000 events were observed in the region that corresponded to the parasites.

StatiStical analySiS
The IC 50 (inhibitory concentration for 50% of cells) CC 50 (cytotoxic concentration for 50% of cells) were calculated by probit analysis (software SPSS ® 13.0), and the selectivity index (SI) were obtained by the ratio between cytotoxicity (CC 50 ) of host cells/IC 50 of promastigotes and amastigotes in macrophages.For the flow cytometry, the graphs were plotted by using the software BD CellQuest software v 6.1.3.The others analyzes, presented results are the mean ± SEM and were analyzed using a one-way analysis of variance, followed by Bonferroni's test for multiple comparisons, for which is p<0.05 was considered significant.

ResuLts and dIscussIon
The major finding of this study is the ethanol extract of stem barks from Z. rhoifolium (ZR-EEtOH) and its n-hexane fraction (ZR-FHEX) presented an effective inhibitory effect of infection of macrophages by L. amazonensis.The ZR-EEtOH fractioning provided the non-polar fraction ZR-FHEX which possesses around 4.0-fold higher antileishmanial activity than ZR-EEtOH against promastigote forms of L. amazonensis (Table I).Interestingly, Moura-Costa et al. (2012) has previously demonstrated the leishmanicidal effect of an aqueous and two hydroalcoholic extracts from Z. rhoifolium stem barks.The addition of ethanol instead of solely water in the extractive process not only enhances the leishmanicidal effect of Z. rhoifolium, but also significantly decreased the cytotoxicity against VERO cells.These findings reinforce evidences of the non-polar fractions as source of compounds and biomarkers for Z. rhoifolium-induced antileishmanial effect.
In this sense, terpenes are non-polar compounds with recognized antileishmanial property (Arruda et al. 2005, Cechinel-Filho andYunes 1998).Mechanisms underlying terpenes-induced antileishmanial effect have been associated to inhibition of protease activity, lipid synthesis, cell cycle or indirectly by modulating macrophage activation (Soares et al. 2012).Terpenes such as nerolidol, an oxygenated sesquiterpene, effectively inhibit isoprenoid biosynthesis, such as dolichol, ergosterol, and ubiquinone in promastigotes (Arruda et al. 2005, Rodrigues et al. 2013).The triterpene dihydrobetulinic acid induces apoptosis-like cell death of L. donovani by targeting DNA topoisomerases (both I and II) and preventing DNA cleavage (Alakurtti et al. 2010).Additionally, the diterpene dolabelladienetriol purified from the marine algae Moreover, the presence of triterpenes in Z. rhoifolium was previously confirmed by qualitative tests (Freitas et al. 2011), as well as by the isolation and identification of lupeol as the major constituent of ZR-FHEX, confirmed by 1 H and 13 C NMR spectroscopic analyses (Pereira et al. 2010).This compound exhibited numerous biological activities including anti-inflammatory, anti-cancerous, cardioprotective, hepatoprotective, anti-microbial and antiprotozoal properties.Considered as a promising antiprotozoal agent, lupeol has demonstrated a significant inhibitory effect against parasites which cause malaria, trypanosomiasis or leishmaniasis (Siddique and Saleem 2011).Accordingly, lupeol demonstrated significant antileishmanial effect against promastigotes of L. braziliensis, L. amazonensis and L. donovani at IC 90 of 100 µg/mL (Fournet et al. 1992).Interestingly, the effect of lupeol obtained from ZR-FHEX against promastigote forms was effective with IC 50 of 13.61 µg/mL at 72 h (data not shown), which might indicates lupeol as a biologically active biomarker of Z. rhoifolium.
The cytotoxicity was evaluated by determination of mean cytotoxic concentration (CC 50 ) against murine peritoneal macrophages by MTT test.The cytotoxicity against macrophages and promastigotes were compared by determination of the selectivity index (SI) (CC 50 /IC 50 ) (Table I), and the results demonstrate toxicological selectivity of ZR-EEtOH and ZR-FHEX to promastigotes rather than macrophages, a valuable characteristic of effective drugs for the treatment of leishmaniasis.Accordingly, Moura-Costa et al. (2012) has demonstrated low cytotoxicity for Z. rhoifolium hydroalcoholic extract in VERO cells, despite of its marked antileishmanial activity, reinforcing the higher selectivity of ZR-EEtOH and ZR-FHEX to the parasites.Interestingly, lupeol-rich extracts and fractions has demonstrated a marked antileishmanial effect and low cytotoxicity.A previous study reported that the hexanic fraction of Brazilian brown propolis possess lupeol-high content and a marked antileishmanial activity against promastigote and amastigote forms of L. amazonensis, as well as low cytotoxicity when compared with the ethanol extract (Santana et al. 2014).
Considering these evidences, the effect of the pretreatment of the macrophages with the ZR-EE-tOH or ZR-FHEX on the resistance to infection by L. amazonensis was also investigated.Three hours after induction of infective process, a significant reduction of infection and number of intracellular amastigote forms was observed in ZR-EEtOH-or ZR-FHEX-pretreated macrophages (Fig. 1).Moreover, the pretreatment of promastigotes with the ZR-EEtOH or ZR-FHEX at concentrations of half the IC 50 at 24 h promoted a significant reduction in the number of internalized amastigotes per macrophage allied to the reduction of the percentage of infected macrophages.The best result was obtained for ZR-FHEX at a concentration around 4.4-fold lower than ZR-EEtOH (Fig. 2).
The activation of macrophages is involved in the control of intracellular infection by parasites from Leishmania genus (Ghazanfari et al. 2006).Accordingly, many antimicrobial enzymatic systems are activated during phagocytosis in order to digest pathogens and/or phagocytized substances, and they probably underlying the increase of cell resistance against Leishmania (Toledo et al. 2009).In this work, a significant increase of phagocytosis activity of zymosan particles was observed in ZR-EEtOH-or ZR-FHEX-pretreated macrophages (Fig. 3a and 3b).Besides, no significant alterations were observed in the determination the lysosomal volume (data not shown).These evidences demonstrate the activation of phagocytic cells probably underlies the Z. rhoifolium-induced antileishmanial effect.
The synthesis and release of nitric oxide (NO) is considered the most effective mechanism under-lying the cell defense of macrophages against parasites from Leishmania genus (Green et al. 1990, Bogdan andRöllinghoff 1998).The parasite is able to survive inside macrophages due to its ability of inhibit the expression or activity of inducible nitric oxide synthase (iNOS), the production of cytokines involved in the regulation of iNOS and the NO synthesis by glycosylinositol phospholipids on the surface of amastigotes, as well as stimulate the production of transforming growth factor TGF-β (Balestieri et al. 2002, Barral-Netto et al. 1992, Proudfoot et al. 1995).In this work, a slight increase of nitrite production was observed after the pretreatment of macrophages with the ZR-EEtOH or ZR-FHEX (Fig. 3c and 3d).Interestingly, ZR-EEtOH and ZR-FHEX promoted a marked increase of nitrite production in macrophages previously infected with promastigotes (data not shown).These findings suggest the induction the Th1 immune response triggered by NO signaling pathway the as a possible underlying mechanism in ZR-EEtOH-or ZR-FHEX-induced antileishmanial effect (Bogdan and Röllinghoff 1998).
In order to evaluate the DNA damage as a possible mechanism underlying antileishmanial effect of Z. rhoifolium against L. amazonensis, promastigote forms were pretreated with ZR-EEtOH or ZR-FHEX at concentrations of 7.96, 15.92 and 31.84µg/mL for 24 h.Then, a marked concentrationdependent decrease of DNA content was observed after pretreatment with ZR-EEtOH or ZR-FHEX, showing a higher efficacy for ZR-FHEX (Fig. 4).Furthermore, after submitting the extracted DNA  to enzymatic digestion in the presence of restriction endonucleases enzymes Eco RI and Hind III, differences between DNA-related bands were not observed when compared with control (data not shown).These results indicate that ZR-EEtOH or ZR-FHEX was not able to change possible sites of action of these endonucleases and then induce mutations in the Leishmania genome.
Then, considering marked decreases in DNA content after exposure to ZR-EEtOH and ZR-FHEX were observed at higher concentrations (15.92 and 31.84μg/mL), ZR-EEtOH-or ZR-FHEX-pretreated promastigote forms of L. amazonensis at concentration of 7.96 μg/mL were submitted to morphological and quantitative analyses by flow cytometry.Scatterplots were detected by the  voltage pulse as a function of the sum of the pulse heights in 10,000 cells for the control group ZR-EEtOH-or ZR-FHEX-pretreated promastigotes, and results were expressed as amplitude (FSC-A) versus area (FSC-W).A decrease in cell size of L. amazonensis promastigote forms was observed after pretreatment with ZR-EEtOH or ZR-FHEX when compared with control group (Fig. 5).Besides, the density of the cell culture was analyzed by the frequency of population and the average of emitted fluorescence, and a decrease of size and number of parasites population, as shown in Fig. 5. Furthermore, the numbers of events were counted for control and each concentration, and a marked reduction was observed in 90.31% and 90.38% for ZR-EEtOH and ZR-FHEX, respectively (Fig. 5).These findings might suggest induction of cell death or inhibition of parasites growth as a possible mechanism underlying ZR-EEtOH-or ZR-FHEX-induced antileishmanial effect.Hence, ZR-EEtOH-and ZR-FHEX-induced antileishmanial effect involves the decrease of the infection and the infectivity rates of macrophages by L. amazonensis.The NO release and the macrophages activation is suggested as probable underlying mechanisms, as well as the inhibition of parasites growth.Moreover, the ZR-FHEX is more effective than ZR-EEtOH considering its better antileishmanial effect and decrease of DNA content of promastigotes.Therefore, further investigation regarding the promising treatment of leishmaniasis by Z. rhoifolium and further underlying mechanisms is markedly reinforced.
morPHology and quantification of L. amazonensis by flow cytometry after Pretreatment witH Z. rhoifolium

Figure 1 -
Figure 1 -Effect of pretreatment of macrophages with ZR-EEtOH or ZR-FHEX, expressed by percentage of macrophages infection (£) and average number of amastigotes per infected macrophage (¢).Values are expressed as mean  s.e.m.One-way ANOVA followed by Bonferroni's post-test; *p<0.05 compared with control.

Figure 3 -
Figure 3 -Phagocytosis of zymosan particles (a and b) and Colorimetric measurement of nitrite (c and d) produced by macrophages pretreated with ZR-EEtOH or ZR-FHEX.Values are expressed as mean absorbance  s.e.m at 550 nm.One-way ANOVA followed by Bonferroni's post-test; **p<0.01,***p<0.001compared with control.LPS: lipopolysaccharide from Escherichia coli.

Figure 4 -
Figure 4 -Quantification of DNA extracted from Leishmania amazonensis after pretreatment with ZR-EEtOH or ZR-FHEX for 72 h.Values are expressed as mean  s.e.m.One-way ANOVA followed by Bonferroni's post-test; ***p<0.001compared with control.

taBLe I Mean inhibitory concentration (Ic 50 ) and mean cytotoxic concentration (cc 50 ) of ethanol extract of stem barks from Z. rhoifolium (ZR-eetoH) and its hexanic fraction (ZR-FHeX) against promastigote forms of L. amazonensis.
Dictyotapfaffii decreases the infection of macrophages by Leishmania spp., even in the presence of factors that exacerbate parasite growth, such asIL-10 and TGF-β (Soares et al. 2012).
a Confidence intervals.BERNARDO MELO NETO et al.